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Inauguraldissertation Charakterisierung plasmamembrangebundener Proteasen von Nicotiana tabacum und Hordeum vulgare Inauguraldissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Manuela Eick geboren am 05. August 1979 in Königs Wusterhausen Greifswald, 21. August 2012 Dekan: Prof. Dr. Klaus Fesser 1. Gutachter: Prof. Dr. Christine Stöhr 2. Gutachter: Prof. Dr. Doris Rentsch Tag der Promotion: 23. November 2012 Inhaltsverzeichnis 1. Einleitung _____________________________________________________________ 1 1.1 Proteasen _______________________________________________________________ 1 1.1.1 Allgemeine Funktionen proteolytischer Enzyme in Pflanzen _______________________________ 1 1.1.2 Klassifizierung von Proteasen _______________________________________________________ 2 1.1.3 Regulationsmechanismen der pflanzlichen Proteaseaktivität _______________________________ 6 1.2 Membrangebundene Proteasen in Pflanzen ______________________________________ 8 1.3 Die pflanzliche Plasmamembran _______________________________________________ 9 1.4 Fragestellungen der Arbeit ___________________________________________________ 11 2. Material und Methoden _________________________________________________ 13 2.1 Material ___________________________________________________________________ 13 2.1.1 Pflanzen ______________________________________________________________________ 13 2.1.2 Sand _________________________________________________________________________ 13 2.1.3 Feinchemikalien ________________________________________________________________ 13 2.1.4 Geräte ________________________________________________________________________ 14 2.1.5 Software ______________________________________________________________________ 15 2.1.6 Wasseraufbereitung _____________________________________________________________ 15 2.2 Methoden _________________________________________________________________ 16 2.2.1 Pflanzenanzucht und Kultivierung von Tabak __________________________________________ 16 2.2.2 Pflanzenanzucht und Kultivierung von Gerste _________________________________________ 17 a) Pflanzenanzucht ___________________________________________________________________ 17 b) Optimierung der Pflanzenanzucht von Gerste ____________________________________________ 18 2.2.3 Pigmentbestimmung der Gerste ____________________________________________________ 19 2.2.4 Exsudatgewinnung und chromatographische Aufarbeitung _______________________________ 20 2.2.5 Präparation der Membranvesikeln __________________________________________________ 20 a) Präparation von mikrosomalen Membranfraktionen (mF) __________________________________ 20 b) Präparation isolierter Plasmamembranvesikel ___________________________________________ 21 2.2.6 Proteinbestimmung ______________________________________________________________ 23 2.2.7 Proteaseaktivitätsbestimmung ______________________________________________________ 24 a) mit Fluoresceinthiocarbamoyl-Casein __________________________________________________ 24 b) mit Exopeptidasesubstraten __________________________________________________________ 26 2.2.8 Endogene Proteolyse an PM-Vesikeln _______________________________________________ 27 2.2.9 Proteinfällung __________________________________________________________________ 28 a) Fällung löslicher Proteine mit Trichloressigsäure ________________________________________ 28 b) Chloroform/Methanol-Fällung solubilisierter PM-Proteine _________________________________ 29 2.2.10 Solubilisierung PM-gebundener Proteine ____________________________________________ 29 2.2.11 Probenwaschung und Volumeneinengung mittels Membranfiltration ______________________ 30 2.2.12 Chromatographische Reinigung der Protease ________________________________________ 30 a) Grössenausschlusschromatographie ___________________________________________________ 30 b) Anionenaustauschchromatographie ___________________________________________________ 31 c) Kationenaustauschchromatographie ___________________________________________________ 32 d) Hydrophobe Interaktionschromatographie ______________________________________________ 32 e) Metallchelat-Affinitätschromatographie ________________________________________________ 33 2.2.13 Behandlung mit Peptid-N-glykosidase F (PNGase F) __________________________________ 34 2.2.14 Gelelektrophorese ______________________________________________________________ 35 a) Native PAGE _____________________________________________________________________ 35 b) SDS-PAGE _______________________________________________________________________ 35 c) Zymogramm ______________________________________________________________________ 36 2.2.15 Silberfärbung _________________________________________________________________ 37 2.2.16 Massenspektrometrie ___________________________________________________________ 38 a) Bearbeitung und Messung solubilisierter PM-Proteine ____________________________________ 38 b) Einfluss der verwendeten Detergenzien _________________________________________________ 39 II.b c) Bearbeitung und Messung der angereinigten PM-Protease Hv-Prot 5mM ______________________ 40 d) Messung der über endogene Proteolyse abgespaltenen Polypeptide __________________________ 42 e) Informationen zu detektierten Proteinen _________________________________________________ 42 3. Ergebnisse ___________________________________________________________ 43 3.1 Charakterisierung membranassoziierter Proteasen _______________________________ 43 a) anhand ihrer Substratspezifitiät _______________________________________________________ 43 b) in Abhängigkeit von der Stickstoffernährung _____________________________________________ 45 3.2 Eine Vielzahl unterschiedlicher Proteasen ist an die Plasmamembran von Wurzeln gebunden _____________________________________________________________________ 46 3.2.1 Untersuchungen zur Bindung der Proteasen an die Plasmamembran in Abhängigkeit von der Nitraternährung _____________________________________________________________________ 46 3.2.2 Fraktionierung der PM-Proteasen mittels chromatographischer Verfahren ___________________ 48 a) molekulare Grössenunterschiede innerhalb solubilisierter PM-Proteasen ______________________ 48 b) Trennung solubilisierter PM-Proteasen aufgrund der Nettooberflächenladung __________________ 49 I II c) Fraktionierung der Prot und Prot anhand der Hydrophobizität ______________________________ 50 I II d) Fraktionierung der Prot und Prot mittels Affinitätschromatographie _________________________ 51 3.2.3 Fraktionierung der PM-Proteasen mittels Gelelektrophorese ______________________________ 52 a) Native PAGE solubilisierter PM-Proteasen ______________________________________________ 52 II.b b) Untersuchung der angereinigter Hv-Prot 5mM mittels SDS-PAGE ____________________________ 55 c) Untersuchung der verschiedenen Proteaseformen mittels Zymographie ________________________ 57 I 3.2.4 Deglykosylierung verändert das Proteasespektrum der Hv-Prot 5mM _________________________ 60 3.3 Verschiedene Proteasetypen sind an die PM von Wurzeln gebunden_________________ 61 3.3.1 Einfluss spezifischer Proteaseinhibitoren _____________________________________________ 61 3.3.2 Einfluss zweiwertiger Kationen _____________________________________________________ 66 3.3.3 Massenspektrometrische Analyse verschiedener Proteaseformen von Gerste __________________ 69 a) Analyse solubilisierter PM-Proteine von Gerstewurzeln ____________________________________ 69 II b) Analyse der fraktionierten Hv-Prot 5mM _________________________________________________ 72 3.4 Natürliche Substrate PM-gebundener Proteasen _________________________________ 80 3.4.1 Untersuchungen zur Substratspezifität der PM-gebundenen Proteasen ______________________ 80 3.4.2 Proteolytisch aktive Proteine werden endogen von den PM-Vesikeln abgespalten ______________ 88 4. Diskussion ___________________________________________________________ 91 4.1 Vielfalt der Proteaseformen an der pflanzlichen Plasmamembran ___________________ 91 a) Metalloproteasen __________________________________________________________________ 93 b) Serinproteinasen __________________________________________________________________ 100 c) Cysteinproteinasen ________________________________________________________________ 101 d) Aspartatproteinasen _______________________________________________________________ 104 e) Hinweise auf weitere, nicht klassifizierbare PM-gebundene Proteasen ________________________ 106 4.2 Kritische Betrachtung der verwendeten Methoden und Resultate __________________ 108 a) Herausforderungen bei der Anreinigung bisher unbekannter Proteasen _______________________ 108 b) Massenspektrometrie nicht-abundanter PM-gebundener Proteasen __________________________ 113 4.3 Wie sind die PM-Proteasen an die Membran gebunden? _________________________ 120 4.4 Physiologische Bedeutung PM-gebundener Proteasen bei Pflanzen _________________________ 126 a) Hinweise zur Regulation der PM-Proteaseaktivität _______________________________________ 126 b) Einfluss physiologischer Faktoren auf die PM-Proteaseaktivitäten ___________________________ 127 c) Funktionen PM-gebundener Proteasen in Wurzeln _______________________________________ 130 d) PM-Proteasen: ein erstes Modell _____________________________________________________ 137 5. Zusammenfassung ___________________________________________________ 138 6. Literatur ____________________________________________________________
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